Organ transplantation is a life-saving procedure for patients with end-stage organ failure. Conventional immunosuppression leads to high morbidity and is unable to prevent chronic transplant dysfunction. The blockade of positive T costimulatory pathways remains amongst the most promising strategies to induce tolerance, although this has yet to be successfully translated into effective clinical protocols. The efficacy of costimulation blockade is diminished by several factors, including initial high frequency of na[unreadable]ve donor-reactive T cell precursors, their potential subsequent differentiation to graft-destructive Th1/Th2/Th17 or protective Tregs (depending on the type of co-stimulatory signal received and the nature of the cytokine milieu in which this occurs), increased B cell responses and the presence of donor-reactive memory T cells in recipients. The recent discovery of [unreadable]negative[unreadable] costimulation pathways is a new and promising area of reseach in the development of tolerance-inducing strategies. Based on preliminary and published data from our group and others, TIM-3:gal-9 is a unique and multifaceted pathway that plays a broad role in down-modulating inflammatory responses in alloimmunity: 1. In addition to triggering cell death in Th1 T cells, TIM-3 is a central master switch that can broadly modulate CD4+ effector T cell differentiation by regulating the production of IL-6 by CD4+ T cells. 2. TIM-3 is expressed by some innate immune cells, and by influencing their function, may affect both innate and adaptive immunity. 3. Galectin-9, the ligand for TIM-3, is widely expressed and may enable parenchymal cells to directly participate in regulation of the alloimmune response. 4. Donor-reactive memory T cells can precipitate costimulatory blockade-resistant allograft rejection;recent evidence, mainly from models of viral infection, suggests an important role of TIM-3:gal-9 pathway in T cell exhaustion and generation and recall of memory responses. 5. Gal-9 deficiency prevents induction of tolerance by CTLA4-Ig, by causing cellular and severe antibody-mediated rejection with myocardial necrosis. This unique model of cellular and humoral cardiac rejection will allow investigation of key cellular and molecular mechanisms by which T cell help is delivered to naive B cells. Such studies have heretofore been difficult to perform due to the lack of experimental mice models. 6. Identification of ATF-3 as a candidate transcription factor that may mediate the anti-inflammatory, and possibly the pro-apoptotic effects of the TIM-3:gal-9 pathway is both novel and promising. 7. The availability of agents that can enhance the inhibitory function of this pathway and prolong allograft survival is indeed novel, and of high clinical significance. The main goal of this proposal is to define the functions and exact cellular and molecular mechanisms of action of the TIM-3:gal-9 pathway in regulating alloimmune responses in vivo as a means of developing novel strategies to achieve durable and reproducible tolerance. Utilization of unique tools combined with close collaboration of the PI with a team of renowned experts will assure the successful completion of this proposal.